The present invention relates to a genomic gene encoding HM1.24 antigen protein, a promoter of the gene encoding HM1.24 antigen protein, and uses thereof.
Mouse anti-HM1.24 monoclonal antibody has been prepared using a human myeloma cell line KPC-32 as an immunogen (Goto, T. et al., Blood 84: 1922-1930, 1994). The HM1.24 antigen that is recognized by this antibody is a membrane protein having a molecular weight of 29 to 33 kDa that is overexpressed on the surface of myeloma cells. Furthermore, for normal cells its expression has been confirmed in immunoglobulin-producing B cells (plasma cells, lymphoplasmacitoide cells), but expression is rarely observed in the other cells and tissues (Goto T. et al., supra). However, nothing is known about HM1.24 antigen except its expression distribution and molecular weight.
According to the present invention, as a result of the cloning of genomic DNA encoding HM1.24 antigen, the determination of its nucleotide sequence and the deduced amino acid sequence, and further homology search, HM1.24 antigen was demonstrated to be a molecule identical with BST2 that is a surface antigen expressed on the stroma cells isolated from the bone marrow of patients with myeloma, and the bone marrow and the snynovial membrane of patients with rheumatoid arthritis. BST2 has been shown to have an ability of supporting the growth of pre-B cells and is thought to be involved in the pathology of rheumatoid arthritis, but its other physiological functions are not known (Ishikawa J. et al., Genomics 26: 527-534, 1995).
In the production of a useful gene product derived from an animal by means of genetic engineering, it often happens that the gene is not expressed, a gene product, protein, does not take a correct conformation, post-translational modification does not occur correctly and the like, when a microorganism host such as Escherichia coli, Bacillus subtilis, or yeast is used. In order to solve such problems, animal cells are often used as hosts, in which case, the selection of a promoter has a great impact on expression efficiency. Conventional, frequently used promoters for animal cells include SV40 promoter, cytomegalovirus promoter, actin promoter, and the like.
Considering the above state of art, the present invention provides a genomic DNA encoding HM1.24 antigen protein.
The present invention also provides a process for producing HM1.24 antigen protein using animal cells by means of said genomic DNA.
When a useful gene product is to be produced in large quantities using animal cells as a host, conventionally used promoters for animal cells are not always satisfactory in terms of transcription activity, and hence there is a great need for the development of stronger promoters. Thus, it is an object of the present invention to provide a DNA having a stronger promoter activity as a promoter for animal cells and uses thereof.
In order to solve the above problems, the present invention provides a genomic DNA encoding HM1.24 antigen protein, said DNA comprising 4 exon regions encoding the amino acid sequence as set forth in SEQ ID NO: 2. As an example of the above genomic DNA, the present invention provides a genomic DNA having 4 exons encoding the amino acid sequence and 3 introns as set forth in SEQ ID NO: 2.
The present invention also provides a splicing variant of the above genomic DNA. Specific examples include a splicing variant lacking exon 2, a splicing variant lacking exons 2 and 3, and the like.
The present invention also provides a process for producing HM1.24 antigen protein which method comprises culturing animal cells transformed with an expression vector comprising the above genomic DNA.
The present invention further provides a promoter sequence DNA having the nucleotide sequence of the 5xe2x80x2-non-coding region as set forth in SEQ ID NO: 4 or a DNA fragment of said sequence having a promoter activity in animal cells.
The present invention also provides a DNA that hybridizes with the above DNA or a fragment thereof under a stringent condition and that has a promoter activity in animal cells. The above DNA having promoter activity is preferably derived from animal cells, in particular mammalian cells.
The present invention also provides a DNA that has been modified by the deletion, addition and/or substitution with other nucleotides, of one or a plurality of nucleotides in the nucleotide sequence of the 5xe2x80x2-non-coding region as set forth in SEQ ID NO: 4 and that has a promoter activity in animal cells.
The present invention also provides a recombinant DNA wherein a useful gene is operably linked to the above DNA having a promoter activity. As the above useful genes, there can be mentioned, for example, nucleic acids selected from the group consisting of nucleic acids encoding useful proteins, antisense DNA, antisense RNA, nucleic acids encoding decoys, and ribozyme.
The present invention also provides a vector comprising the above recombinant DNA. The vector is a plasmid vector or a virus vector.
The present invention also provides animal cells into which the above recombinant DNA has been introduced.
The present invention also provides animal cells that have been transformed with the above vector.
The present invention also provides an animal having the above animal cells.
The present invention also provides a method of expressing a useful gene which method comprises culturing animal cells into which the above recombinant DNA has been introduced.
The present invention also a process for producing a useful protein which process comprises sulturing animal cells transformed with an expression vector comprising a nucleic acid encoding a useful protein operably linked to the above DNA having a promoter activity.